Cells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part, by increasing glucose-derived histone acetylation. We recently expanded the known metabolites that integrate into epigenetic signaling and found that medium-chain lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. This signaling axis leads to activation of stress response genes, including lipid metabolic genes, and inhibition of cell cycle gene expression. Our preliminary data presented in this application expand upon these gene expression observations and reveal lipid oxidation induces potent suppression of the cell cycle, lowers cellular proliferation across a range of cell lines, and lowers tumor burden in a mouse model of liver cancer. However, a critical gap in our knowledge remains that prevent us from fully understanding how the role of lipids and their oxidation in cancer: In which sub-cellular organelle are medium-chain lipids oxidized to generate acetyl-CoA for histone acetylation? Is the oxidation of MCTs required for their effect on cellular proliferation? What are the key cellular mediators of these epigenetic signals? There is a critical need to understand the full range of lipid?s molecular effects in order to enable more intelligent therapies for cancer. Although it is widely accepted that mitochondria oxidize lipids and are prime candidate sites of mediating communication between nutrients and the nucleus, no data exist to support this notion. Therefore, the single objective of this exploratory grant application is to determine the site(s) and molecular mediators of lipid signaling on epigenetics. Aim 1 will test three candidate sites of lipid metabolism using a combination of pharmacological and genetic approaches. In a complementary parallel approach, Aim 2 will perform an unbiased CRISPR- Cas9 screen to identify genes that are required for the suppressive effects of medium-chain lipids on cellular proliferation. This project is significant because it will explain the molecular mechanisms by which lipids and their oxidation mediate the positive effects cellular proliferation and cancer outcomes. We put forth conceptual and technical innovations that will both follow directed hypotheses and allow unbiased discovery of the most important aspects of the response to medium-chain lipids. Successful completion of this project will identify new aspects of inter- organelle communication in cancer.